Method and device for polymerase chain reaction

ABSTRACT

An apparatus for amplifying a nucleic acid sequence in a polymerase chain reaction (PCR) is provided. The apparatus comprises a microprocessor configured to communicatively couple with a user input device and receive instructions from the user input device and implement the received instructions; an electromagnetic radiation (EMR) generator configured to generated EMR at frequencies ranging from 300 THz to 400 THz and communicatively coupled with the microprocessor; a reaction vessel, communicatively coupled with the microprocessor, to house the nucleic acid sequence, necessary reagents to the PCR, and particles comprising a transition metal material, and to receive the EMR generated by the EMR generator; and a temperature sensor communicatively coupled with the reaction vessel and the microprocessor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. non-provisional patentapplication Ser. No. 14/840,833, filed on Aug. 31, 2015, which claimsthe benefit of U.S. provisional patent application No. 62/044,413, filedon Sep. 2, 2014, the entirety of which are incorporated herein byreference.

FIELD

The present disclosure relates to amplifying nucleic acid sequences bypolymerase chain reaction (PCR). More particularly, the presentdisclosure relates to amplifying nucleic acid sequences in PCR by use ofelectromagnetic radiation (EMR) in combination with EMR frequencyabsorbing particles having a transition metal material as a heatingsource.

BACKGROUND

Polymerase chain reaction (PCR) is a technique discovered to synthesizemultiple copies of one or more fragments of DNA from a single ormultiple target templates. The original PCR process is based on heatstable DNA polymerase enzyme from Thermus aquaticus (Taq), which cansynthesize a complimentary strand of a given DNA strand in a mixturecontaining the four types of DNA bases (cytosine, guanine, adenine, andthymine) and a pair of DNA primers, each primer at a terminus of thetarget DNA sequence. The mixture is heated to separate the double helixDNA into individual strands containing the target DNA sequence and thencooled to allow the primers to hybridize with their complimentarysequences on the separate strands and the Taq polymerase to extend theprimers into new complimentary strands. Repeated heating and coolingcycles multiply the target DNA exponentially, for each newly formeddouble helix separates to become two templates for further synthesis.

A typical temperature profile for the polymerase chain reactionincludes, (1) denaturation at 95° C. for 15 to 30 seconds; (2)hybridization with primers at appropriate annealing temperature for 30to 60 seconds; and (3) elongation or extension of the hybridized primersat 72° C. for a period of time depending on the length of the DNA to beamplified, typically for about 30 to 60 seconds. The denaturation andhybridization Its steps occur almost instantly, however, in conventionalPCR apparatus, the temperature changes at a rate approximately 1°C./second when a metal heating block or water is used for thermalequilibration. This conventional thermal cycle is inefficient for itrequires heating and cooling of material other than the DNA sampleitself.

BRIEF DESCRIPTION OF THE DRAWINGS

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings, where:

FIG. 1 is a diagram illustrating the structures of an exemplary mini PCRdevice and its interaction with a user device in accordance with aspectsof the present disclosure;

FIG. 2 is diagram illustrating an exemplary interface between the miniPCR device and the user device as depicted in FIG. 1 in accordance withaspects of the present disclosure;

FIG. 3A illustrates the EMR frequency absorption profile of Fe₃O₄nanoparticles at terahertz wavelengths in accordance with aspects of thepresent disclosure;

FIG. 3B illustrates the EMR frequency absorption profile of the Fe₃O₄nanoparticles at 1.3 MHz in accordance with aspects of the presentdisclosure;

FIG. 4A is the time course of temperature changes of the particles ofexample 1.1 in accordance with aspects of the present disclosure;

FIG. 4B illustrates the rate of increasing the temperature of theparticles of example 1.1 during PCR cycle in accordance with aspects ofthe present disclosure;

FIG. 4C illustrates the rate of decreasing the temperature of theparticles of example 1.1 during PCR cycle in accordance with aspects ofthe present disclosure;

FIG. 5A illustrates the variation in temperature of various types ofnanoparticles along with time, in which the concentration of thenanoparticles is 1,250 ppm in accordance with aspects of the presentdisclosure;

FIG. 5B illustrates the variation in temperature of various types ofnanoparticles along with time, in which the concentration of thenanoparticles is 2,500 ppm in accordance with aspects of the presentdisclosure; and

FIG. 6 illustrates the result of a PCR in accordance with aspects of thepresent disclosure.

DETAILED DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

Procedures of PCR involve a great deal of heating at controlledtemperatures, which is not only tedious but also time and energyconsuming. In the present disclosure, by use of electromagneticradiation (EMR), in combination with EMR absorbing particles having atransition metal material, as the heating source, it is possible toelevate the temperature of a PCR reaction mixture at a rate of about13-15° C./sec, thereby allowing the PCR to proceed in a much faster andmore energy efficient manner in a miniature device. Further, rapidtransitions from one temperature to another ensure that the sample(e.g., target DNA sequence) spends a minimum amount of time atundesirable intermediate temperatures to optimize the fidelity andpurity of the amplified DNA.

The present disclosure is directed to a method for amplifying a nucleicacid sequence of a reaction mixture in a polymerase chain reaction(PCR). The method includes contacting the reaction mixture withparticles formed from a material having a transition metal, a nitride, aphosphide or an arsenide of a Group III metal doped with the transitionmetal, or silicon dioxide doped with the transition metal. The methodcan further include irradiating the particles with electromagneticradiation (EMR) having a frequency of about 200 kilohertz (kHz) to 500terahertz (THz), such that the target nucleic acid sequence isamplified. The Group III metal can be any one of aluminum (Al), gallium(Ga), and indium (In). The transition metal can be any one of manganese(Mn), iron (Fe), cobalt (Co), and copper (Cu). The temperature of thereaction mixture in the PCR can be increased at a rate of about 13-15°C./sec and decreased at a rate of about 6-7° C./sec.

The present disclosure further is directed to a method for amplifying anucleic acid sequence of a reaction mixture in a polymerase chainreaction (PCR). The method includes contacting the reaction mixture withparticles having a material having a transition metal oxide, atransition metal hydroxide, a silicon dioxide doped with the transitionmetal oxide, or a silicon dioxide doped with the transition metalhydroxide. The method can further include irradiating the particles withelectromagnetic radiation (EMR) having a frequency of about 200 kHz to500 THz, such that the target nucleic acid sequence is amplified. Thetransition metal, the transition metal oxide or transition metalhydroxide can be composed, in whole or in part, of one of manganese(Mn), iron (Fe), cobalt (Co), and copper (Cu). The temperature of thereaction mixture in the PCR is increased at a rate of about 13-15°C./sec and decreased at a rate of about 6-7° C./sec. According to one ormore embodiments of the present disclosure, the transition metal oxideor hydroxide can be any one or more of FeO, Fe₂O₃, Fe₃O₄, FeO(OH),Fe(OH)₂, Fe(OH)₃, MnO, Mn₃O₄, Mn₂O₃, MnO(OH), MnO₂, CoO, CoO(OH), Co₃O₄,and CuO.

According to one or more embodiments, the particle can be any one ormore of FeO, Fe₂O₃, or Fe₃O₄.

According to one or more embodiments, the nitride, phosphide or arsenideof Group III metal doped with the transition metal or metal ionincludes, but is not limited to, gallium nitride (GaN) doped with Mn,Fe, Co or Cu; aluminum nitride (AlN) doped with Mn, Fe, CO or Cu, or anion thereof aluminum phosphide (AlP) doped with Fe, Co or Cu, or an ionthereof; indium phosphide (InP) doped with Fe, Co or Cu, or an ionthereof; gallium arsenide (GaAs) doped with Fe, Co or Cu, or an ionthereof, and indium arsenide (InAs) doped with Fe, Co or Cu, or an ionthereof.

The step of irradiating the particles comprises increasing thetemperature of the reaction mixture to a first temperature rangerequired for denaturing DNA, typically about 80 to 105° C. for about 0.5to 1 minute; increasing the temperature of the reaction mixture to asecond temperature range for primer pair annealing, typically about 35to 65° C. for about 0.5 to 1 minute so that a pair of primers hybridizewith the denatured target nucleic acid sequence; and increasing thetemperature of the reaction mixture to a third temperature range ofpolymerase activity, typically about 40 to 80° C., for about 0.5 to 5minutes depending on the length of the target amplified sequence, sothat the target nucleic acid sequence is amplified by elongationsynthesis.

In some embodiments, the particles are directly mixed with the targetnucleic acid in the reaction mixture, and each particle in the reactionmixture has a hydrodynamic diameter of about 100 to 800 nm.

In other embodiments, the reaction mixture is held in a vessel composedof the particles having a transition metal material. For example, insome embodiments one or more films, coatings, or layers having theparticles described above, can be disposed on a surface of the vessel.The one or more films, coatings or layers can each be composed of one ormore monolayers of the panicles described above.

The one or more films, coatings, or layers can be fabricatedindependently of the vessel and then disposed on the vessel. Forexample, particle-containing thin films can be formed in a matrix orcarrier material can be formed and subsequently disposed onto a surfaceof the vessel by any one of hot, cold, or solvent lamination techniques.One of ordinary skill will recognize that any method of disposing theone or more films, coatings, or layers that results in strong adherenceto the vessel surface, thereby preventing detachment or delaminationfrom the vessel surface, is highly desirable.

The one or more films, coatings, or layers can be formed directly on theon the vessel. The one or more films, coatings, or layers can be formeddirectly on the on the vessel by any one of, chemical vapor deposition(CND), physical vapor deposition, spray coating, brushing, dip coating,or any other suitable method. One of ordinary skill will recognize thatany method of directly forming the one or more films, coatings, orlayers that results in strong adherence to the vessel surface, therebypreventing detachment or delamination from the vessel surface, is highlydesirable.

Accordingly, the present disclosure also encompasses a device foramplifying a target nucleic acid sequence of a reaction mixture in aPCR. The device includes a tube or container holder, or a reactionvessel, configured to allow the reaction mixture to indirectly ordirectly contact the particle of the present disclosure; an EMRfrequency generator configured to emit radiation having frequency ofabout 200 kHz to 600 THz, which is subsequently directed to the sampleholder or reaction vessel for irradiating the particles; and amicroprocessor coupled with the sample holder or the reaction vessel, afan, and the EMR frequency generator, for increasing and decreasing thetemperatures of the reaction mixture respectively at a rate of about13-15° C./sec and 6-7° C./sec.

As described above, the generated EMR frequency can be in a range of 200kHz to 600 THz. Alternatively, the generated EMR frequency can be in arange of 100 THz to 600 THz, alternatively 200 THz to 500 THz, andalternatively, 300 THz to 400 THz.

As described above, the device includes a reaction vessel for holdingthe reaction mixture; and a temperature sensor coupled with the vessel,to detect the temperature of the reaction mixture. The device furtherincludes an EMR frequency generator. The device further includes atemperature control circuit for controlling the temperature sensor.Additionally, the device can include a control circuit configured tomodulate the intensity of the EMR frequency generated from the EMRfrequency generator. In at least one, implementation, the device canalso include a microprocessor coupled with the temperature controlcircuit and the heat control circuit. The microprocessor can beconfigured to increase the temperature of the reaction mixture to afirst range of about 80 to 105° C. for about 0.5 to 1 minutes todenature the target nucleic acid sequence. The microprocessor canfurther be configured to increase the temperature of the reactionmixture to a second temperature range of about the annealing temperatureof the primers, typically in a range of 35 to 65° C. for about 0.5 to 1minute, to hybridize a pair of DNA primers with the denatured targetnucleic acid sequence. The microprocessor can be configured to increasethe temperature of the reaction mixture to a third temperature range ofabout 40 to 80° C. for about 0.5 to 1 minute, depending on the length ofthe target sequence, to amplify the target nucleic acid sequence byelongation synthesis.

The device can further comprise a wireless communication circuit such asa BLUETOOTH®, a ZIGBEE® or a WIFI™ circuit for wirelessly communicatingwith the user input device via sending and receiving information to andfrom the device.

According to at least one embodiment, the device is about 300 to 500 cm³or less in size.

Accordingly, one aspect of the present disclosure is to provide a methodfor amplifying a target nucleic acid sequence of a reaction mixture in aPCR. The method includes contacting the reaction mixture with EMRfrequency absorbing particles formed from a material that is atransition metal, a nitride, a phosphide or an arsenide of a Group IIImetal doped with the transition metal or a transition metal ion, orsilicon dioxide doped with the transition metal. The method can furtherinclude irradiating the EMR absorbing particles with EMR having afrequency of about 200 kHz to 500 THz to amplify the target nucleic acidsequence. The Group III metal is any one of Al, Ga, and In. Thetransition metal is any one of Mn, Fe, Co and the transition metal ionis an ion of any one of Mn, Fe, Co and Cu. The temperatures of thereaction mixture in the PCR are raised and cooled respectively at a rateof about 13-15° C./sec and 6-7° C./sec.

Another aspect of the present disclosure is to provide a method foramplifying a target nucleic acid sequence of a reaction mixture in aPCR. The method includes contacting the reaction mixture with EMRfrequency absorbing particles formed from a material that is atransition metal oxide or transition metal hydroxide, or silicon dioxidedoped with the transition metal oxide or transition metal hydroxide; andirradiating the EMR absorbing particles with EMR having a frequency ofabout 200 kHz to 500 THz to amplify the target nucleic acid sequence.The transition metal oxide or hydroxide can comprise any one of Mn, Fe,Co and Cu. The temperatures of the reaction mixture in the PCR areraised and cooled respectively at a rate of about 13-15° C./sec and 6-7°C./sec.

In general, any particles capable of absorbing and releasing heat (e.g.,originated from light or EMR frequency) to its surrounding area can beused as the heating source, in the method of the present disclosure.

The particles that are a transition metal oxide or transition metalhydroxide include, but are not limited to, FeO, Fe₂O₃, Fe₃O₄, FeO(OH),Fe(OH)₂, Fe(OH)₃, MnO, Mn₃O₄, Mn₂O₃, MnO(OH), MnO₂, CoO, CoO(OH), Co₃O₄,and CuO. In at least one example, the particles include FeO, Fe₂O₃, orFe₃O₄.

The particles that are nitrides, phosphides or arsenides of a Group IIImetal doped with the transition metal or a transition metal ion include,but are not limited to, GaN doped with Mn, Fe, Co or Cu, or an ionthereof; AlN doped with Mn, Fe, Co or Cu, or an ion thereof; AlP dopedwith Fe, Co or Cu; MP doped with Fe, Co or Cu, or an ion thereof; GaAsdoped with Fe, Co or Cu, or an ion thereof; and InAs doped with Fe, Coor Cu, or an ion thereof.

Each type of particles can have similar or unique energy conversionproperties. According; to exemplary embodiments of the presentdisclosure, the EMR absorbing; particles are iron oxide particlesselected from the group consisting of FeO, Fe₂O₃, and Fe₃O₄. In at leastone example, iron oxide particles of the present disclosure exhibitstrong absorption to EMR frequencies of about 300 to 400 THz,particularly at 371 THz. In another example, the iron oxide particlesexhibit strong absorption to EMR having frequencies of 200 kHz to 2 MHz,particularly, at 1.3 megahertz (MHz). Due to the high heat conversionefficiency of the iron oxide particles of the present disclosure, thetemperature of the particles after being irradiated with the EMR canincrease at a rate of about 13-15° C./sec. In addition, the iron oxideparticles also allow fast cooling when EMR is turned off, with a coolingrate of about 6-7° C./sec.

The iron oxide particles of the present disclosure can be about 10 to1,200 nm in diameter; alternatively, about 50 to 1,000 nm in diameter;alternatively about 80 to 800 nm in diameter. In one example, twopreparations of iron oxide particles respectively having different sizesare prepared.

One preparation of the iron oxide particles results in particlesrelatively small in size, with each particle being about 60 to 150 nm indiameter; such as 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 nm indiameter; alternatively about 80 to 120 nm in diameter, such as 80, 90,100, 110 or 120 nm; alternatively about 100 nm in diameter. Particleswithin the 60 to 150 nm diameter range have been found to exhibitdesired dispersion properties in solvents and resulting layers,coatings, or films having requisite magnetic properties for EMRabsorption.

The second preparation of the iron oxide particles result in particlesrelatively large in size with each particle being about 200 to 1,200 nmin diameter; such as 200, 300, 400, 500, 600, 700, 800, 900, 1,000,1,100 or 1,200 nm in diameter; alternatively about 400 to 1,000 nm indiameter, such as 400, 500, 600, 700, 800, 900 or 1,000 nm;alternatively about 800 nm in diameter. Particles within the 200 to1,200 nm diameter range have been found to exhibit superior magneticproperties over the 60 to 150 nm diameter range particles with reduceddispersion in solvents.

According to embodiments of the present disclosure, the iron oxideparticles of the present disclosure, either small or large in size,exhibit good energy conversion properties, with the large size ironoxide particles exhibiting the best energy conversion properties tobring the temperature of a PCR mixture to about 100° C. within 15 sec.

One approach of employing iron oxide particles as the heating source ina PCR reaction is bye directly adding a sufficient amount of the ironoxide particles to the PCR reaction mixture, and then proceed with thePCR reactions as normal.

Alternatively, the iron oxide particles of the present disclosure can beformed into a vessel, as described above, for receiving the PCR mixturedirectly or indirectly therein. The term “directly” as used hereinrefers to loading the PCR mixture into or onto the vessel formed by amaterial containing the iron oxide particles of the present disclosure.The term “indirectly” means the PCR mixture is loaded into a container(e.g., an Eppendorf tube) before being placed into the vessel formed bya material containing the iron oxide particles of the presentdisclosure. In at least one exemplary embodiment, the iron oxideparticles of the present disclosure are formed into a microfluidicbiochip reaction chamber, in which the PCR reaction mixture is loadeddirectly into the reaction chamber or coated on the reaction chamber. Inanother exemplary embodiment, the iron oxide particles of the presentdisclosure are formed into a thin film, on which various reaction spots,each containing a PCR reaction mixture are placed, and each of the PCRreactions proceed simultaneously on the film.

FIG. 1 is a diagram of an exemplary device 100 of the present disclosurefor conducting PCR as described herein. In the exemplary embodiment, thedevice 100 is wirelessly controlled by an application program in a userinput device 102 (for example, a mobile phone, laptop computer, tabletcomputer, or other similar device). The device 100 is about 9 cm(length)×8 cm (width)×5 cm (height) in size or less.

FIG. 2 is diagram illustrating an exemplary interface between the miniPCR device and the user device as depicted in FIG. 1. To start to PCRprocedure, the target DNA intended to be amplified, the necessaryreagents (e.g., Taq, the four types of nucleotides, the DNA primerpairs, and a buffer solution and the particles of the present disclosureare loaded into a reaction tube or container (e.g., an Eppendorf tube),which is then placed into a tube or container holder 124, and the holder124 is subsequently placed in a reaction vessel 126. Alternatively, thetarget DNA and the necessary reagents are loaded directly into thevessel 126, in which the vessel 126 is composed of a material comprisingthe particles of the present disclosure.

A PCR user can start the process by entering all the necessaryparameters (for example, temperature and time of the reaction) requiredfor running a PCR into an application program installed in a hand-helddevice, such as the user input device 102 exemplified as a mobile phonein FIG. 1. The information is then sent from the user input device 102to a wireless communication circuit 118. A microprocessor 114, uponreceiving the necessary parameters from the wireless communicationcircuit 118, then coordinates a temperature control circuit 112 and aheat control circuit 116 to execute the parameters by instructing an EMRfrequency generator 132 to emit a suitable EMR frequency, and atemperature sensor 122 to initiate the requested PCR. Specifically, theEMR frequency emitted from the EMR frequency generator 132 is directedto the reaction tube or container that is held in the holder 124, or tothe vessel 126, thereby raising the temperature of the PCR reactionmixture therein to the designated temperature. The EMR frequency shallmatch the energy conversion properties of the particles in the PCRreaction mixture held in the holder 124, or the particles contained inthe reaction vessel 126. For example, in the case when iron oxideparticles of the present disclosure are used, an EMR frequency of about371 THz can be used.

Whether the PCR reaction mixture has reached its designated temperatureis measured and determined by the temperature sensor 122, which is underthe control of the temperature control circuit 112 via themicroprocessor 114. After the PCR reaction mixture has reached thedesignated temperature and been held at that temperature for adesignated time, the microprocessor 114 will then control the heatcontrol circuit 116, to instruct the EMR frequency generator 132 to stopgenerating the EMR frequency; and the temperature sensor 122, to actuatea fan 128 to prevent the PCR mixture from over-heating or to start thecooling cycle. In other words, during the heating cycle, themicroprocessor 114 coordinates the heat control circuit 116 and thetemperature control circuit 112 to respectively instruct the EMRfrequency generator 132 to generate EMR and the fan 128 to run or notrun, depending on the desired heating rate of the PCR procedure.Similarly, during the cooling cycle, the microprocessor 114 coordinatesthe heat control circuit 116 and the temperature control circuit 112 toinstruct the EMR frequency generator 132 to stop generating EMR and thefan 128 to run. Since the EMR frequency in combination with theparticles of the present disclosure are used as the heating source, thetemperature of the PCR reaction mixture can increase at a rate of about13-15° C./sec and decrease at a rate of about 6-7° C./sec, therebyallowing the heating and cooling cycles to complete in an energy andtime efficient manner.

In at least one example, amplification of 279 bps target DNA per cycleis completed in less than 60 seconds, which in general will take atleast 180 seconds in a conventional PCR device.

In essence, b using an EMR frequency and EMR frequency absorbingparticles, the present method and/or device significantly reduce thereaction time of a conventional PCR by at least 67%, thereby allowingthe amplification of DNA fragments to be completed in a much more timeand energy efficient manner, without affecting the optimum fidelity andpurity of the amplified DNA product. Further, the present device issmall in size, no more than 500 cm³, and can be remotely activated.Thus, the present device eliminates the need of having the PCR operatorto physically activate the device to start the reaction.

Without being bound to any particular theory, it presently believed thatthe mechanism of nanoparticle excitation may be as follows.Nanoparticles, such as, for example, iron oxide nanoparticles, can besubjected to irradiation using a radiation source having a predefinedfrequency. The radiation has sufficient energy to excite an electron ofan iron atom in an inner orbital or shell of the atom to a higher energyunoccupied orbital or shell. This produces a metal ion with an electronmissing from the inner orbital. The resulting electron configuration isunstable, and an electron from an orbital of higher energy drops intothe half-filled orbital. Energy, which may be in the form of heat, isemitted as a result of the electron dropping into the half-filledorbital.

Alternatively, without being bound to any particular theory, itpresently believed that the mechanism of nanoparticle excitation may beas follows. Nanoparticles, such as, for example, iron oxidenanoparticles, can be subjected to irradiation using a radiation sourcehaving a predefined frequency. The radiation can be absorbed by theatoms of the nanoparticles without ionization of said atoms. Theabsorbed radiation, rather, results in excitation and increasedvibration of intermolecular bonds of the nanoparticles, generating heatwhich is released to areas surrounding the nanoparticles.

The present disclosure will now be described more specifically withreference to the following embodiments, which are provided for thepurpose of demonstration rather than limitation.

EXAMPLES

Materials and Methods

PCR reaction mixture. The PCR reaction mixture contained the followings:5 pmol of primer (SEQ ID NO: 1,5′-gcgaaagtcctggttgagctgag-3′; SEQ ID NO:2,5′-aacccaaggcccatgcataca-3′), 1 μg template DNA, polymerase, PCRbuffer, bovine serum albumin (BSA), nucleotides, and deionized water.The total volume was 20 μL.

PCR procedures. The PCR was conducted according to the followingtemperature profiles: (1) denaturation at 95° C. for 15 to 30 seconds;(2) hybridization with primers at 56° C. for 15 to 30 seconds; and (3)elongation or extension of the hybridized primers at 72° C. for 15 to 30seconds.

Example 1 Preparation and Characterization of Particles

1.1 Preparation of Iron Oxide (Fe₃O₄) Particles

Briefly, FeCl₂ (10 mL and 50 mM), Trimesic acid (4.5 mL and 25 mM),citrate (0.15 g), NaOH (18 mg), and N₂H₂ (100 μL) and gelatin were mixedand reacted at 155° C. for 12 hrs. The size and shape of the thusproduced Fe₃O₄ nanoparticles were then determined by TransmissionElectron Microscopy (TEM) and Scanning Electron Microscopy (SEM).Relatively small and large sizes Fe₃O₄ nanoparticles respectively havingan average diameter of about 103±43 nm and about 828±559 nm wereproduced.

The produced Fe₃O₄ particles were found to absorb EMR at two differentfrequency ranges, one within the terahertz range (FIG. 3A, maximumabsorption occurred at about 371 THz), the other within the megahertzrange (FIG. 3B, 1.3 MHz). The Fe₃O₄ particles were then subjected toheating and cooling cycles commonly employed in PCR by irradiating theparticles with EM frequency of 371 THz, the time course of temperaturechanges is illustrated in FIG. 4A, whereas the respective heating andcooling rates of the particles of examples 1.1 are depicted in FIGS. 4Band 4C. It was found that the particles of examples 1.1 has an averageheating rate of 13.93±0.65° C./sec (FIG. 4B), and an average coolingrate of 6.39±0.50° C./sec (FIG. 4C).

With respect to the average heating rate, after multiple experimentsusing the same experimental parameters, it was found that the particlesof example 1.1 exhibited a maximum heating rate of 14.7° C./sec, aminimum heating rate of 13.0° C./sec, a median heating rate of 13.9°C./sec, a heating rate of 13.3° C./sec in a first quartile ofexperiments, and a heating rate of 14.5° C./sec in a third quartile ofexperiments (See FIG. 4B).

With respect to the average cooling rate, after multiple experimentsusing the same experimental parameters, it was found that the particlesof example 1.1 exhibited a maximum cooling rate of 7.24° C./sec, aminimum cooling heating rate of 5084° C./sec, a median cooling rate of6.17° C./sec, a cooling rate of 5.95° C./sec in a first quartile ofexperiments, and a cooling rate of 6.80° C./sec in a third quartile ofexperiments (See FIG. 4C).

1.2 Preparation of Gold Nanorod Particles

The Au nanorods were produced and characterized by a method similar toexample 1.1. The synthesis of Au nanorods followed a procedure similarto that described by B. Nikoobakht and M. A. El-Sayed, Chem. Mater.2003, 15, 1957-1962. Briefly, a seed solution (12 μL) containsHexadecyltrimethylammoniumbromide (CTAB) (5 ml, 0.20 M) and HAuCl₄ (5ml, 0.0005 M) is added to 10 mL of growth solution which contains (CTAB)(5 ml, 0.20 M) and AgNO₃ (5 ml, 0.004 M). The reaction is kept constantat room temperature. The size of the produced Au nanorods was thendetermined by TEM. Each of the produced Au nanorods was about 45 nm inlength, and 10 nm in width.

1.3 Characterization of the Photo-Thermal Properties of the Particles ofExamples 1.1, 1.2 and Carbon Nanotubes (CNTs)

In this example, the photo-thermal properties of the particles ofexamples 1.1 were compared with other particles, including the goldnanorods of example 1.2 and carbon nanotubes (CNTs) (Golden InnovationBusiness Co., Ltd. Taiwan), by subjecting different concentrations ofparticles (1,250 or 2,500 ppm) to EMR emitted from 250, 500 or 1,000milliwatt (mW) light emitting diodes (LEDs), and temperature changesover time were measured. Results are illustrated in FIGS. 5A and 5B.

It was found that the respective temperatures of Fe₃O₄ particles, Aunanorods and CNTs were slightly above room temperature (i.e., about 29°C.) (See FIG. 5A) upon being irradiated with an EMR frequency generator(250 mW, 371 THz). The temperatures increased to about 60° C. for smallsize Fe₃O₄ particles (ranging from about 50 nm to about 200 nm) and Aunanorods, about 65° C. for CNTs, and about 79° C. for large size Fe₃O₄particles (ranging from about 250 nm to about 1400 nm) when the power ofthe EMR frequency generator was elevated to 500 nm. When the power wasraised to 1,000 mW, then the temperature increased to about 85° C. forsmall size Fe₃O₄ nanoparticles and Au nanorods, about 110° C. for CNTs,and about 120° C. for large size Fe₃O₄ nanoparticles. Taken together,the temperature of each type of particles increases in proportion to theincrease in the power of the EMR frequency generator; and among thethree types of particles, Fe₃O₄ particles, particularly the large sizeFe₃O₄ particles, exhibit the best energy conversion properties.

Similar results were observed when the concentration of each type ofparticles were increased to be 2,500 ppm (see FIG. 5B).

Example 2 PCR Conducted Using the Fe₃O₄ Particles of Example 1.1

2.1 PCR Reaction Mixtures Comprising the Fe₃O₄ Particles of Example 1.1

In this example, 1000 ppm of the Fe₃O₄ particles of example 1.1 weredirectly mixed with the PCR reaction mixture in a reaction tube, thetube was then subjected to PCR cycles, by (1) irradiating the tube withradiation frequency of 371 THz emitted from a Laser Diode (700 mW) untilthe temperature reached 95° C., and held (500 mW) for 15 to 30 seconds;(2) activating the fan system to decrease the temperature until itreached 56° C., and held (250 mW) for 15 to 30 seconds; and (c)irradiating the tube again with 371 THz of EMR frequency until thetemperature reached 72° C., and held for 15 to 30 seconds. The PCRcycles were repeated 30 times, and the amplified product was detected byelectrophoresis (FIG. 6, Lane 4). A 100 bp YEA Ladder DNA Marker II(Yeastern Biotech Co., Ltd., Cat. No. FYD009-1ML) was used as areference (FIG. 6, Lane 1).

The above procedure was compared to a conventional PCR procedure (FIG.6, Lane 5). The conventional procedure was performed as follows. First,a reaction mixture containing a DNA sample was heated to 95° C. for 10minutes for denaturation of the DNA sample. Second, the denaturation wasrepeated for 30 cycles, each cycles being performed at 95° C. for 30seconds. Third, hybridization with corresponding DNA primers wasperformed at 56° C. for 30 seconds. Fourth, the hybridized primers wereelongated at 72° C. for 30 seconds. Finally, an additional elongationstep was performed 72° C. for 10 minutes to form the final product asshown in Lane 5 of FIG. 6.

2.2 PCR Conducted in a Vessel Made From the Fe₃O₄ Particles of Example1.1

In this example, PCR cycles are performed by placing the PCR reactionmixtures in a vessel made of a material comprising the Fe₃O₄ particlesof example 1.1. The PCR cycles are then initiated in accordance with thesteps described above.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications can be made bythose with ordinary skill in the art. The above specification, examplesand data provide a complete description of the structure and use ofexemplary embodiments. Although various embodiments have been describedabove with a certain degree of particularity, or with reference to oneor more individual embodiments, those with ordinary skill in the artcould make numerous alterations to the disclosed embodiments withoutdeparting from the spirit or scope of this disclosure.

Statements of the Disclosure

Statement 1: A method for amplifying a nucleic acid sequence in apolymerase chain reaction (PCR), the method comprising contacting, in areaction vessel, a reaction mixture comprising target nucleic acids withparticles comprising a transition metal material; and irradiating theparticles with electromagnetic radiation (EMR) having a frequency ofabout 200 kHz to 500 THz.

Statement 2: The method according to Statement 1 wherein the transitionmetal material comprises a transition metal, a transition metal oxide, atransition metal hydroxide, a Group III metal compound doped with thetransition metal or a transition metal ion, a silicon dioxide doped withthe transition metal, a silicon dioxide doped with the transition metaloxide, or a silicon dioxide doped with the transition metal hydroxide.

Statement 3: The method according to any one of Statements 1 or 2,wherein the temperature of the reaction mixture in the PCR is increasedat a rate of about 13-15° C. per second.

Statement 4: The method according to any one of Statements 1-3, whereinthe temperature of the reaction mixture in the PCR is decreased at arate of about 6-7° C. per second.

Statement 5: The method according to any one of Statements 2-4, whereinthe Group III metal compound is any one of a nitride, a phosphide or anarsenide, and the Group III metal is any one of aluminum (Al), gallium(Ga), and indium (In).

Statement 6: The method according to any one of Statements 1-5, whereinthe transition metal material comprises a combination of one or moretransition metal oxides and transition metal hydroxides.

Statement 7: The method according to any one of Statements 1-6, whereinthe transition metal material comprises one or more of FeO, Fe₂O₃,Fe₃O₄, FeO(OH), Fe(OH)₂, Fe(OH)₃, MnO, Mn₃O₄, Mn₂O₃, MnO(OH), MnO₂, CoO,CoO(OH), Co₃O₄, and CuO.

Statement 8: The method according to any one of Statements 2-6, whereinthe transition metal material comprises any one of manganese (Mn), iron(Fe), cobalt (Co), and copper (Cu).

Statement 9: The method according to any one of Statements 1-8, whereinat least one of the particles has a hydrodynamic diameter of about 10 to1,200 nm.

Statement 10: The method according to any one of Statements 1-8, whereinat least one of the particles has a hydrodynamic diameter of about 200to 1,200 nm.

Statement 11: The method according to any one of Statements 1-8, whereinat least one of the particles has a hydrodynamic diameter of about 60 to150 nm.

Statement 12: The method according to any one of Statements 1-11,wherein irradiating the particles comprises increasing the temperatureof the reaction mixture to a first temperature range of about 80 to 105°C. for about 0.5 to 1 minute, to denature the target DNA; increasing thetemperature of the reaction mixture to a second temperature range ofabout 35 to 65° C. for about 0.5 to 1 minute to hybridize with thedenatured target nucleic acid sequence a pair of primers; and increasingthe temperature of the reaction mixture to a third temperature range ofabout 40 to 80° C., for about 0.5 to 5 minutes, to amplify the targetnucleic acid sequence by elongation synthesis.

Statement 13: An apparatus for amplifying a nucleic acid sequence in apolymerase chain reaction (PCR), the apparatus comprising: amicroprocessor configured to communicatively couple with a user inputdevice and receive instructions from the user input device and implementthe received instructions; a reaction vessel, communicatively coupledwith the microprocessor, to house the nucleic acid sequence, reagentsfor the PCR, and particles; a temperature sensor communicatively coupledwith the reaction vessel and the microprocessor; and an electromagneticradiation (EMR) generator, communicatively coupled with themicroprocessor and configured to direct the EMR toward the reactionvessel, wherein the particles comprise a transition metal material.

Statement 14: The apparatus according to Statement 13, wherein the EMRgenerator is configured to generate EMR at frequencies ranging from 100THz to 600 THz.

Statement 15: The apparatus according to Statement 13, wherein the EMRgenerator is configured to generate EMR at frequencies ranging from 300THz to 400 THz.

Statement 16: The apparatus according to Statement 13, wherein the EMRgenerator is configured to generate EMR at frequencies ranging from 1MHz to 2 MHz.

Statement 17: The apparatus according to any one of Statements 13-16,further comprising a temperature control circuit for controlling thetemperature sensor of the reaction housing; and a heat control circuitfor modulating the intensity of the EMR frequency generated from the EMRfrequency generator, wherein the microprocessor is coupled with thetemperature control circuit and the heat control circuit.

Statement 18: The apparatus according to Statement 17, wherein themicroprocessor is configured to increase the temperature of the reactionmixture to a first temperature range of about 80 to 105° C., for about0.5 to 1 minute, to denature the target DNA; increase the temperature ofthe reaction mixture to a second temperature range of about 35 to 65° C.for about 0.5 to 1 minute to hybridize with the denatured target nucleicacid sequence a pair of primers; and increase the temperature of thereaction mixture to a third temperature range of about 40 to 80° C., forabout 0.5 to 5 minutes, to amplify the target nucleic acid sequence byelongation synthesis.

Statement 19: The apparatus according to any one of Statements 13-18,further comprising a wireless communication circuit for wirelesscommunication with the user input device.

Statement 20: The apparatus according to any one of Statements 13-19,wherein the transition metal material comprises a transition metal, atransition metal oxide, a transition metal hydroxide, a Group III metalcompound doped with the transition metal or a transition metal ion, asilicon dioxide doped with the transition metal, a silicon dioxide dopedwith the transition metal oxide, or a silicon dioxide doped with thetransition metal hydroxide.

Statement 21: The apparatus according to any one of Statements 13-20wherein transition metal material comprises one or more of FeO, Fe₂O₃,Fe₃O₄, FeO(OH), Fe(OH)₂, Fe(OH)₃, MnO, Mn₃O₄, Mn₂O₃, MnO(OH), MnO₂, CoO,CoO(OH), Co₃O₄, and CuO.

Statement 22: The apparatus according to Statement 20, wherein the GroupIII metal compound is any one of a nitride, a phosphide or an arsenide,and the Group III metal is any one of aluminum (Al), gallium (Gal, andindium (In).

Statement 23: The apparatus according to any one of Statements 13-22,wherein the transition metal material comprises a combination of one ormore transition metal oxides and transition metal hydroxides.

Statement 24: The apparatus according to any one of Statements 13-23,wherein the transition metal material comprises any one of manganese(Mn), iron (Fe), cobalt (Co), and copper (Cu).

Statement 25: The apparatus according to any one of Statements 13-24,wherein at least one of the particles has a hydrodynamic diameter ofabout 10 to 1,200 nm.

Statement 26: The apparatus according to any one of Statements 13-24,wherein at least one of the particles has a hydrodynamic diameter ofabout 200 to 1,200 nm.

Statement 27: The apparatus according to any one of Statements 13-24,wherein at least one of the particles has a hydrodynamic diameter ofabout 60 to 150 nm.

Statement 28: The apparatus according to any one of Statements 13-27,wherein the user input device is one of a mobile phone, a laptopcomputer, and a tablet computer.

Statement 29: The apparatus according to any one of Statements 13-28,wherein the apparatus is no more than 500 cm³ in volume.

Statement 30: A polymerase chain reaction (PCR) vessel, the vesselcomprising one or more films, coatings, or layers of nanonarticlescomprising a transition metal material on a surface of the vessel.

Statement 31: The vessel according to Statement 30, wherein thetransition metal material comprises a transition metal, a transitionmetal oxide, a transition metal hydroxide, a Group III metal compounddoped with the transition metal or a transition metal ion, a silicondioxide doped with the transition metal, a silicon dioxide doped withthe transition metal oxide, or a silicon dioxide doped with thetransition metal hydroxide.

Statement 32: The vessel according to any one of Statements 30-31,wherein transition metal material comprises one or more of FeO, Fe₂O₃,Fe₃O₄, FeO(OH), Fe(OH)₂, Fe(OH)₃, MnO, Mn₃O₄, Mn₂O₃, MnO(OH), MnO₂, CoO,CoO(OH), Co₃O₄, and CuO.

Statement 33: The vessel according to Statement 31, wherein the GroupIII metal compound is any one of a nitride, a phosphide or an arsenide,and the Group III metal is any one of aluminum (Al), (Ga), and indium(In).

Statement 34: The vessel according to any one of Statements 30-33,wherein the transition metal material comprises any one of manganese(Mn), iron (Fe), cobalt (Co), and copper (Cu).

Statement 35: The vessel according to any one of Statements 30-35,wherein the nanoparticles comprise a combination one or more transitionmetal oxides and transition metal hydroxides.

Statement 36: The vessel according to any one of Statements 30-35,wherein at least one of the particles has a hydrodynamic diameter ofabout 10 to 1,200 nm.

Statement 37: The vessel according to any one of Statements 30-35,wherein at least one of the particles has a hydrodynamic diameter ofabout 200 to 1,200 nm.

Statement 38: The vessel according to any one of Statements 30-35,wherein at least one of the particles has a hydrodynamic diameter ofabout 60 to 150 nm.

Statement 43: A microfluidic biochip, wherein the microfluidic biochipcomprises a vessel according to any one of statements 30-38.

Statement 44: Nanoparticles for generating heat after irradiated byelectromagnetic radiation (EMR) at frequencies ranging from about 300 to400 THz in a PCR apparatus, the nanoparticles have a hydrodynamicdiameter of about 50 to 200 nm, wherein the nanoparticles comprise atransition metal oxide or a transition metal hydroxide.

The foregoing descriptions of specific compositions and methods of thepresent disclosure have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit thedisclosure to the precise compositions and methods disclosed andobviously many modifications and variations are possible in light of theabove teaching. The examples were chosen and described in order to bestexplain the principles of the disclosure and its practical application,to thereby enable others skilled in the art to best utilize thedisclosure with various modifications as are suited to the particularuse contemplated. It is intended that the scope of the disclosure bedefined by the claims appended hereto and their equivalents.

What is claimed is:
 1. An apparatus for amplifying a nucleic acidsequence in a polymerase chain reaction (PCR), the apparatus comprising:a microprocessor communicatively coupled to a user input device andconfigured to receive instructions from the user input device andimplement the received instructions; an electromagnetic radiation (EMR)generator communicatively coupled to the microprocessor and configuredto generate EMR at frequencies ranging from 300 THz to 400 THz; areaction vessel communicatively coupled to the microprocessor,configured to receive EMR from the EMR generator and to house thenucleic acid sequence; at least one transition metal particle disposedin the reaction vessel accessible to the nucleic acid sequence, whereina material of the transition metal particle includes Fe₃O₄ without Aucoating; and a temperature sensor communicatively coupled to thereaction vessel and the microprocessor.
 2. The apparatus of claim 1,further comprising: a temperature control circuit for controlling thetemperature sensor; and a heat control circuit for modulating theintensity of EMR from the EMR frequency generator, wherein themicroprocessor is coupled to the temperature control circuit and theheat control circuit.
 3. The apparatus of claim 2, wherein themicroprocessor is configured to: cause the temperature of the reactionmixture to a first temperature range of about 80 to 105° C., for about0.5 to 1 minute; cause the temperature of the reaction mixture to asecond temperature range of about 35 to 65° C., for about 0.5 to 1minute; and cause the temperature of the reaction mixture to a thirdtemperature range of about 40 to 80° C., for about 0.5 to 5 minutes. 4.The apparatus of claim 1, further comprising a wireless communicationcircuit for wireless communication with the user input device.
 5. Theapparatus of claim 1, wherein at least one of the transition metalparticles has a hydrodynamic diameter of about 10 to 1,200 nm.
 6. Theapparatus of claim 1, wherein at least one of the transition metalparticles has a hydrodynamic diameter of about 200 to 1,200 nm.
 7. Theapparatus of claim 1, wherein at least one of the transition metalparticles has a hydrodynamic diameter of about 60 to 150 nm.
 8. Theapparatus of claim 1, wherein the user input device is one of a mobilephone, a laptop computer, and a tablet computer.
 9. The apparatus ofclaim 1, wherein the apparatus is no more than 500 cm³ in volume.